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Ana Claudia Fernandez2015.Pdf UNIVERSIDADE ESTADUAL DO OESTE DO PARANÁ PRÓ-REITORIA DE PESQUISA E PÓS-GRADUAÇÃO MESTRADO EM CIÊNCIAS FARMACÊUTICAS ANA CLAUDIA APARECIDA MARIANO FERNANDEZ Avaliação da atividade antioxidante e antibacteriana do extrato bruto e frações das folhas de Tetradenia riparia Hochst. Codd (Lamiaceae) CASCAVEL - PR 2015 ANA CLAUDIA APARECIDA MARIANO FERNANDEZ Avaliação da atividade antioxidante e antibacteriana do extrato bruto e frações das folhas de Tetradenia riparia Hochst. Codd (Lamiaceae) Dissertação apresentada ao Programa de Pós-graduação em Ciências Farmacêuticas (PCF-UNIOESTE) da Universidade Estadual do Oeste do Paraná – UNIOESTE Campus Cascavel, para a obtenção do título de Mestre. Orientador: Prof. Dr. Maurício Ferreira da Rosa Co-orientadora: Profª. Drª. Zilda Cristiani Gazim CASCAVEL - PR 2015 Dados Internacionais de Catalogação-na-Publicação (CIP) F413a Fernandez, Ana Claudia Aparecida Mariano Avaliação da atividade antioxidante e antibacteriana do extrato bruto e frações das folhas de Tetradenia riparia Hochst Codd (Lamiaceae) . / Ana Claudia Aparecida Mariano Fernandez.— Cascavel, 2015. 92 p. Orientador: Prof. Dr. Maurício Ferreira da Rosa Coorientadora: Profª. Drª. Zilda Cristiani Gazim Dissertação (Mestrado) – Universidade Estadual do Oeste do Paraná, Campus de Cascavel, 2015 Programa de Pós-Graduação Stricto Sensu em Ciências Farmacêuticas 1. Plantas Medicinais. 2. Antioxidante. 3. Antimicrobiana. I. Rosa, Maurício Ferreira da. II.Gazim, Zilda Cristiani. III. Universidade Estadual do Oeste do Paraná. IV. Título. CDD 21.ed. 615.32 581.634 CIP-NBR 12899 Ficha catalográfica elaborada por Helena Soterio Bejio – CRB 9ª/965 BIOGRAFIA DA AUTORA Ana Claudia Aparecida Mariano Fernandez filha de Venâncio Fernandez Martinez e Josefina F. Mariano Fernandez, nascida em Umuarama-PR em 14 de outubro de 1991.Graduada em Farmácia Generalista pela Universidade Paranaense – UNIPAR - Campus Umuarama – PR (2009–2013), durante a graduação participou dos projetos de Iniciação Científica (PIC e PIBIC), atuou como Farmacêutica Homeopata para produtos veterinários na Empresa Minerphós Saúde e Nutrição Animal - Umuarama-PR (2014). Atualmente Mestranda no Programa de Pós-Graduação de Ciências Farmacêuticas da Universidade Estadual do Oeste do Paraná – UNIOESTE (2014-2015). v “Você pode sonhar, criar, desenhar e construir o lugar mais maravilhoso do mundo. Mas é necessário ter pessoas para transformar seu sonho em realidade!“ Walt Disney vi Dedico este trabalho а Deus, pela força е coragem durante toda esta longa caminhada. Aos meus pais Venâncio e Josefina, as minhas irmãs Carla e Maria e ao meu amor Glauber. vii AGRADECIMENTOS Agradeço primeiramente a Deus todo-poderoso pelo dom de vida que me concedeu e por ter iluminado o meu caminho durante estes anos, por ter oferecido a oportunidade de viver, evoluir e conhecer as pessoas citadas abaixo. Á pessoa que ainda no ensino fundamental com elogios, sorrisos, dedicação e amor fez de mim uma apaixonada pelos estudos, minha mãe e professora Josefina e ao meu pai Venâncio pelo esforço, paciência e amor incondicional. Essa conquista também é de vocês! A minhas irmãs Carla e Maria pela cumplicidade, parceria, paciência, incentivo e amor que me fizeram seguir em frente durante as dificuldades. Em memória dos meus avós José, Jandira e Oliva exemplos de amor, simplicidade e honestidade. Figuras de grande importância em minha formação e de quem sinto muita saudade. Ao meu avô Antônio que admiro por tamanha força e superação, me fazendo acreditar que a perseverança é capaz de superar as dificuldades. Ao meu amor Glauber por ter me aturado nos momentos de estresse, pela paciência em minha ausência e por fazer parte da minha vida. Te amo Ao Prof. Dr. Maurício Ferreira da Rosa pela oportunidade, paciência, aprendizado, apoio e disponibilidade durante todo o desenvolvimento da dissertação. A Profa. Dra. Zilda CristianeGazim pela co-orientação, suporte, paciência e dedicação. Aos Prof. Giani Linde Colauto, Orlando S. Takemura, Vera L. D. Siqueira que me acompanharam e muito colaboraram na realização desse trabalho. viii A Universidade Estadual do Oeste do Paraná (UNIOESTE)e Universidade Paranaense (UNIPAR) pela viabilização deste trabalho. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e a Fundação Araucária pelo apoio financeiro. A todos que direta e indiretamente fizeram parte da elaboração deste trabalho. ix Avaliação da atividade antioxidante e antibacteriana do extrato bruto e frações das folhas de Tetradenia riparia Hochst. Codd (Lamiaceae) RESUMO Desde os primórdios da humanidade as plantas são utilizadas para o tratamento de enfermidades devido as suas propriedades terapêuticas, pois constituem uma fonte de compostos biologicamente ativos com grande interesse científico para as indústrias farmacêutica, cosmética e alimentícia. Hoje, a busca por produtos benéficos ao homem e ao meio ambiente tem aumentado a pesquisa na área antioxidante e antimicrobiana, devido ao relato de toxicidade e resistência. Desta forma, este trabalho teve o intuito de avaliar a atividade antioxidante e antimicrobiana do extrato bruto e das frações de Tetradenia riparia. A ação antioxidante foi avaliada pelos métodos sequestro do DPPH(2,2-difenil-1-picril-hidrazila),sistema β-caroteno/ ácido linoleico, método de redução do ferro (FRAP) e potencial antioxidante reativo total (TRAP). O fracionamento cromatográfico em coluna proporcionou a obtenção de 10 frações, que destas a fração obtida a partir da eluição com acetato de etila: metanol (8:2)(FR-IX) apresentou resultados promissores quanto ao potencial -1 -1 antioxidante com 181,67 µg de ácido gálico mg , CI50 de 0,61 µg mL , 55,61% de proteção, 4,59 µM sulfato ferroso mg-1 e 4,15 µM de trolox em 62,5 µg mL-1, respectivamente. Por Ressonância Magnética Nuclear (RMN) na FR-IX foram detectadas a presença da astragalina, luteolina e boronolida.A ação antimicrobiana foi avaliada pelo método de microdiluição em caldo (MIC) com a determinaçãoda concentração inibitóriamínima (CIM), as frações obtidas pelas fases móveisdiclorometano: hexano 7:3 e 8:2 (FR-II); diclorometano: hexano 9:1 e diclorometano 100% (FR-III) apresentaram potencial antibacteriano sobre a cepa ATCC 29213Staphylococcus aureus com CIM de 1,95 e 0,98 µg mL-1, respectivamente. Deste modo, o fracionamento cromatográfico foi de suma importância para detectar a presença de frações com atividade antioxidante e antibacteriana superior ao extrato bruto de mirra. Palavras-chave: plantas medicinais, antioxidante, antimicrobiana. x Evaluation of the antioxidant and antibacterial activity of the crude extract and fractions of the leaves of Tetradenia riparia Hochst. Codd (Lamiaceae) ABSTRACT Since human being earliest days, plants is used to diseases treatment due their therapeutic properties, once they are a source of biologically active compounds with great scientific interest for pharmaceutical, cosmetic and food industries. Nowadays, the pursuit for beneficial products to human and to environment has increased the research on antioxidant and antimicrobial areas due to the toxicity and resistance reports. This work aimed to evaluate antioxidant and antimicrobial activities of crude extract and fractions of Tetradenia riparia. Antioxidant action was evaluated through the DPPH scavenging (2,2-diphenyl- 1-picrylhydrazyl), β-Carotene/ linoleic acid assay, ferric reducing (FRAP) and total reactive antioxidant potential (TRAP). Column chromatographic fractionation provided 10m fractions, where among them the fraction gotten by the elution with ethyl acetate: methanol (8:2) (FR-IX) present promising results -1 in relation to its antioxidant potential with 181,67 µg of gallic acid mg , CI50 of 0,61 µg mL-1, 55,61% of protection, 4,59 µM ferrous sulfate mg-1 and 4,15 µM of trolox in 62,5 µg mL-1, respectively. By nuclear magnetic resonance (NMR) in the FR-IX was detected the presence of astragalin, luteolon and boronolide. Antimicrobial activity was evaluated by broth microdilution method with the determination of the minimum inhibitory concentration (MIC), fraction obtained by dichloromethane mobile phases: hexane 7:3 and 8:2 (FR-II); dichloromethane: hexane 9:1 and dichloromethane 100% (FR-III) presented an antibacterial potential on ATCC 29213 Staphylococcus aureus strains with MIC of 1,95 and 0,98 µg mL-1, respectively. In this way, chromatographic fractioning was utmost important to detect the presence of fractions with superior antioxidant and antibacterial activities to the myrrh crude extract. Keywords: Medicinal plants, antioxidante, antimicrobial xi SUMÁRIO LISTA DE SÍMBOLOS, SIGLAS E ABREVIAÇÕES 14 LISTA DE FIGURAS 15 LISTA DE TABELAS 16 1. INTRODUÇÃO 17 2. OBJETIVOS 18 2.1 Objetivo Geral 18 2.2 Objetivos específicos 18 3. REVISÃO DE LITERATURA 19 3.1 Plantas Medicinais 19 3.2 Metabolismo secundário 20 3.3 Família lamiaceae 22 3.4 Tetradenia riparia (Hochst.) Codd 23 3.5 Antioxidantes 27 3.6 Antimicrobianos 30 4. REFERÊNCIAS 34 ARTIGO - Avaliação da atividade antioxidante e antibacteriana do extrato bruto e frações das folhas de Tetradenia riparia Hochst. Codd (Lamiaceae) RESUMO 42 ABSTRACT 43 INTRODUÇÃO 44 MATERIAIS E MÉTODOS 45 Material vegetal e coleta 45 Obtenção do extrato hidroalcoólico bruto de T. riparia 46 Fracionamento do extrato bruto 46 Identificação química 47 Atividade antioxidante 47 Quantificação de fenóis totais 47 Sequestro dos radicais livres do DPPH 47 Método sistema beta-caroteno/ácido linoleico 48 Método de redução do ferro (FRAP) 49 xii Potencial antioxidante reativo (TRAP)
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